Ectopic hCG-beta secreted by DoT and CaSki human carcinoma cell lines contains two components, ectopic hCG-beta-I and ectopic hCG-beta-II. Ectopic hCG-beta-II lacks the C-terminal peptide (CTP), based on lack of recognition by antibodies against CTP, lack of susceptibility to thermolysin (which normally cleaves the CTP), and lack of binding of the desialylated derivative to Arachis hypogaea (AH) lectin (specific for Gal-GalNAc of the O-linked oligosaccharides on the CTP). Immunoreactive hCG-beta-like material has been detected in the serum or urine of 13 of 18 patients with cervical carcinoma. Conditions for large-scale roller bottle culture of CaSki cervical carcinoma cells were optimized. A method to separate ectopic hCG-beta-I from ectopic hCG-beta-II has been validated and involves differential affinity chromatography with: (1)\anti-hCG-alpha Sepharose; (2)\anti-[CTP-directed]hCG-beta-Sepharose; and (3)\anti-[core-directed]hCG-beta Sepharose. Ectopic hCG-beta-II is eluted from the third stage with 4 M guanidine\HC1, pH 3, and desalted. The purified ectopic hCG-beta-II failed to combine with standard hCG-alpha under conditions where standard hCG-beta combined with standard hCG-alpha. Evidence was obtained for the presence of additional carbohydrates on ectopic hCG-beta. DoT and CaSki culture fluids contained immunoreactive hCG-beta-like components of apparently larger size than standard hCG-beta (from pregnancy urine hCG) as demonstrated by gel filtration and sodium dodecyl sulfate-gel electrophoresis (the latter following incubation of CaSki cells with radioactive amino acids). (C)